Studies will be conducted on the replication of the plasmid RK2 to understand the basis for its broad host-range. Temperature-sensitive replication mutants will be isolated and characterized by complementation with genes cloned into other plasmids. Heteroduplex structures of P plasmids and cloned DNA fragments will reveal the organization of these genes. Additional mutants will be generated by insertion of a transposon. Both sets of mutants, and deletions of RK2 previously generated by restriction endonucleases, will be tested for altered host-range, incompatibility and plasmid copy number. Properties of the origin/terminus replication complex will be investigated by studies of molecules diploid for this region. Electron microscopic examination of replicative intermediates will indicate if both origins are active, and if a termination signal exists. The basis for the suppression of the Col E1 replicon in hybrid plasmids of covalently linked Col E1 and RK2 will be investigated by studies of composite plasmids of Col E1 and temperature-sensitive replication mutants of RK2. Mutants of RK2-Col E1 hybrids, able to replicate in chloramphenicol-treated cultures, will also be selected, and replicative intermediates scanned to determine the location of the Col E1 replication block. Construction of an in vitro system for RK2 replication will be initiated to test the function of isolated proteins implicated in the replication of P plasmids. Such proteins will be identified by transfer of plasmids containing the cloned genes into minicells of E. coli, and the proteins synthesized in this background characterized physically.